1. A preparation method of a phospholipid bilayer membrane for nanopore sequencing is characterized by sequentially comprising the following steps:

(1) mixing hydrophobic small molecular monomer, photoinitiator and phospholipid molecules to obtain a phospholipid mixture;

(2) smearing the phospholipid mixture on the nanopore substrate, and volatilizing the solvent to obtain a phospholipid membrane;

(3) and adding an ionic buffer solution into bath pools on two sides of the nanopore base material to redissolve phospholipid molecules, then recoating a phospholipid mixture on the nanopore base material, and carrying out photoinitiated polymerization to obtain the phospholipid bilayer membrane for nanopore sequencing.

2. The method for preparing the phospholipid bilayer membrane for nanopore sequencing according to claim 1, wherein in the step (1), the molar ratio of the hydrophobic small molecule monomer to the phospholipid molecule is 1: 1-10.

3. The method for preparing the phospholipid bilayer membrane for nanopore sequencing according to claim 1, wherein in the step (1), the molar ratio of the hydrophobic small molecule monomer to the photoinitiator is 1-10: 1.

4. The method of preparing the phospholipid bilayer membrane for nanopore sequencing according to claim 1, wherein in the step (1), the hydrophobic small molecule monomer is at least one of 2-ethylhexyl methacrylate, triethylene glycol dimethacrylate, hydroxyethyl methacrylate, hydroxyethyl acrylate, N-diethylacrylamide, propyl 3- (dimethylamino) acrylate, glycidyl methacrylate, and 2-hydroxypropyl methacrylamide.

5. The method of claim 1, wherein in the step (1), the photoinitiator is 2, 2-diethoxy-1-phenylhexanone, a-methylphenyl mercaptobenzophenone, or trimethylformyldiphenylphosphine oxide.

6. The method for preparing a phospholipid bilayer membrane for nanopore sequencing according to claim 1, wherein in step (2), the phospholipid molecule is diphytanoylphosphatidylcholine.

7. The method for preparing the phospholipid bilayer membrane for nanopore sequencing according to claim 1, wherein in the step (2), the nanopore substrate is prepared by the following method: selecting polystyrene or polycarbonate as a substrate material, carrying out SU-8 pretreatment, and then carrying out epoxy ring-opening activation by using perfluorooctyl trichlorosilane as a modified material to obtain the nanopore substrate.

8. The method according to claim 1, wherein in the step (3), the ionic buffer solution is a mixture of 0.1M potassium chloride solution and 5mM HEPES solution, and the pH value is 7.4.

9. The method for preparing the phospholipid bilayer membrane for nanopore sequencing as claimed in claim 1, wherein in the step (3), the phospholipid bilayer membrane is irradiated for 10min-2h under the ultraviolet light with the wavelength of 254-365nm and the power of 5-50W at the irradiation temperature of 4-37 ℃ during the photo-initiated polymerization.

10. The phospholipid bilayer membrane for nanopore sequencing prepared by the method for preparing a phospholipid bilayer membrane for nanopore sequencing according to any one of claims 1-9.

Background

DNA is the code and blueprint that make up the life. The sequence of bases in nucleotides in DNA constitutes genetic information that can be transcribed to form RNA, which is then translated to produce a polypeptide, which forms a protein. DNA methylation is used as a relatively stable modification state, can be inherited to new filial generation DNA along with the DNA replication process under the action of DNA methyltransferase, and is an important epigenetic mechanism; therefore, 5-methylcytosine, the only methylated form in mammals, is also called the "fifth base" of humans, and abnormal DNA methylation is closely related to the occurrence and progression of various diseases. Therefore, accurate detection of the base arrangement sequence and the methylation condition thereof on human DNA provides strong evidence for human prediction of various diseases.

Gene sequencing technology is a new technology capable of determining the sequence of biomolecules such as nucleic acids or amino acids. Nowadays, although the sequencing instrument is iterated for several times, the first three generations of technologies all have important defects which are difficult to overcome, so that a fourth generation of gene sequencing instrument is promoted to meet higher requirements of human beings on gene sequencing. The fourth generation gene sequencing technology is also called nanopore sequencing technology, and the principle is that under the action of electric field force, different basic groups generate different current signals when single-stranded nucleic acid molecules pass through a nanopore channel, and the DNA sequence distribution in the nucleic acid molecules can be identified by observing the difference of the electric signals. Compared with the previous three-generation sequencing technology, the fourth-generation sequencing technology is a sequencing method which really realizes the combination of single-molecule detection and electronic conduction detection, has ultrahigh read length, high flux, less sequencing time and simpler data analysis, and realizes the double crossing from low read length to ultrahigh read length and from optical detection to electronic conduction detection. However, the artificial double-layer phospholipid membrane is an indispensable part in the nanopore sequencing technology, and at present, a preparation method which can be stably repeated still does not exist, and the market progress of a fourth-generation gene sequencer is seriously slowed down. Therefore, the development of a preparation method of a novel artificial bilayer membrane with high repeatability and stability is an invention with great technical significance and market significance.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a phospholipid bilayer membrane for nanopore sequencing and a preparation method thereof, which are used for solving the problems of poor stability, difficulty in repeated preparation and the like of the phospholipid bilayer membrane in the prior art by doping a hydrophobic micromolecule monomer with double bonds with phospholipid and carrying out in-situ polymerization.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows: the preparation method of the phospholipid bilayer membrane for nanopore sequencing comprises the following steps in sequence:

(1) mixing hydrophobic small molecular monomer, photoinitiator and phospholipid molecules to obtain a phospholipid mixture;

(2) smearing the phospholipid mixture on the nanopore substrate, and volatilizing the solvent to obtain a phospholipid membrane;

(3) and adding an ionic buffer solution into bath pools on two sides of the nanopore base material to redissolve phospholipid molecules, then recoating a phospholipid mixture on the nanopore base material, and carrying out photoinitiated polymerization to obtain the phospholipid bilayer membrane for nanopore sequencing.

Furthermore, in the step (1), the mole ratio of the hydrophobic small molecular monomer to the phospholipid molecule is 1: 1-10.

Further, in the step (1), the mole ratio of the hydrophobic small molecular monomer to the photoinitiator is 1-10: 1.

Further, in the step (1), the hydrophobic small molecule monomer is at least one of 2-ethylhexyl methacrylate, triethylene glycol dimethacrylate, hydroxyethyl methacrylate, hydroxyethyl acrylate, N-diethylacrylamide, 3- (dimethylamino) propyl acrylate, glycidyl methacrylate and 2-hydroxypropyl methacrylamide.

Further, in the step (1), the photoinitiator is 2, 2-diethoxy-1-phenylhexanone, alpha-methyl phenyl mercapto benzophenone or trimethylformyl diphenyl phosphine oxide.

Further, in the step (2), the phospholipid molecule is diphytanoylphosphatidylcholine.

Further, in the step (2), solvent evaporation is performed by standing evaporation or nitrogen evaporation.

Further, in the step (2), the nanopore substrate is prepared by the following method: selecting polystyrene or polycarbonate as a substrate material, carrying out SU-8 pretreatment, and then carrying out epoxy ring-opening activation by using perfluorooctyl trichlorosilane as a modified material to obtain the nanopore substrate.

Further, in the step (3), the ionic buffer was a mixture of 0.1M potassium chloride solution and 5mM HEPES solution at pH 7.4.

Further, in the step (3), during photo-initiated polymerization, the polymer is irradiated for 10min-2h under ultraviolet light with the wavelength of 254-365nm and the power of 5-50W, and the irradiation temperature is 4-37 ℃.

Further, the phospholipid membranes prepared should include the following classes:

solid diphytylphosphatidylcholine was dried with argon gas and then vacuumed overnight, and then dissolved in n-decane, and conventional non-polymeric phospholipid bilayer membranes (BLMs) were prepared from this solution.

Further, a hydrophobic polymerizable small molecule monomer is doped into a phospholipid solution, and a mixture of DPhPC (diphytylphosphatidylcholine), the hydrophobic polymerizable small molecule monomer and a photoinitiator is used to prepare mixed non-polymerized phospholipid bilayer membranes (MA-BLMs) without uv polymerization.

Further, polymer monomers, a photoinitiator and a DPhPC solution are mixed to form a film on the nano-pores, and the film is irradiated and crosslinked by ultraviolet light to prepare the polymerized phospholipid double-layer films (PS-BLMs).

Further, the specific steps for preparing the MA-BLMs and the PS-BLMs are as follows:

removing a free radical inhibitor in a polymer monomer by using an alumina column, and mixing the free radical inhibitor with a photoinitiator DEAP according to a certain proportion to obtain a monomer mixture;

adding DPhPC with a certain proportion into the monomer mixture, stirring for 5min to obtain a monomer-doped phospholipid mixture, and dissolving the monomer-doped phospholipid mixture into n-decane, wherein the final phospholipid concentration is 20 mg/mL;

smearing 2 mu L of phospholipid or phospholipid/monomer mixed solution on the nano-pores, and drying by using nitrogen gas to form BLM;

adding 1mL buffer solution (1M KCl, 5mM HEPES, pH 7.4) into the bath pool at two sides of the nanopore to redissolve the phospholipid or phospholipid/monomer mixture, and connecting the phospholipid or phospholipid/monomer mixture to a reference electrode through a salt bridge;

sucking a trace amount of phospholipid or a phospholipid solution doped with hydrophobic polymerizable micromolecules by using a pipette gun, and repeatedly and lightly sweeping the periphery of the nanopore to form a membrane; the formation of BLM was determined by monitoring the increase in resistance across the nanopore.

Further, a phospholipid bilayer internal polymer stabilizing scaffold can be formed by:

s1: ultraviolet irradiation is carried out by an ultraviolet lamp at a position 1-2cm away from the BLM, so that the monomer-doped phospholipid mixture is subjected to photocrosslinking, and finally the PS-BLM is formed;

s2: the stability of BLMs is characterized by measuring breakdown voltage and lifetime. The breakdown voltage value is the potential at which irreversible rupture of the BLMs occurs, and the breakdown voltage value of the phospholipid membrane is obtained by applying increasing potentials from 0 to 2000mV in increments of 10mV for 50ms, the average breakdown voltage value reflecting the electrical stability of the BLM. The phospholipid membrane life refers to the average time required for the double-layer membrane to break under the action of +/-5 mV 20Hz square waves;

s3: selecting gramicidin A (Grancidin A) or hemolysin (alpha-HL) as transmembrane protein, adding 2 mu L of alpha-HL solution (1mg/mL) into a bath pool with 1mL of buffer solution at one side of a nanopore, embedding the protein into a phospholipid membrane under the bias potential of +40mV, and monitoring the change of resistance at two sides of the nanopore to determine whether the protein is successfully embedded.

The phospholipid bilayer membrane for nanopore sequencing is prepared according to the preparation method of the phospholipid bilayer membrane for nanopore sequencing.

In summary, the invention has the following advantages:

1. the invention utilizes the hydrophobic micromolecule monomer with double bonds to be doped with phospholipid and carry out in-situ polymerization, so as to solve the problems of poor stability, difficult repeated preparation and the like of the phospholipid double-layer membrane in the prior art. In the preparation process, hydrophobic small molecules, an initiator and diphytanoylphosphatidylcholine (DPhPC) without polymerization capacity are mixed, and the small molecule monomers have hydrophobicity, so the small molecule monomers can be gathered in a tail foot sheet layer of a lipid bilayer, and then the phospholipid membrane is subjected to in-situ polymerization by ultraviolet irradiation, so that the artificial bilayer membrane with high stability and high ion channel activity is obtained.

2. Polystyrene or polycarbonate is selected as a base material, and SU-8 pretreatment is performed on the base material, so that a nonpolar surface with a water contact angle of about 85 degrees is provided for the base material, and the method is more suitable for forming a lipid bilayer. Perfluorooctyl trichlorosilane is selected as a modified material, the SU-8 pretreated base material is subjected to epoxy ring-opening activation, and then perfluoro compounds are grafted to the base material, so that a modified base with stronger hydrophobicity is obtained, the hydrophilic and hydrophobic effect between the base material and phospholipid molecules is enhanced, and the phospholipid molecules form a film on the surface of the modified base material more stably.

3. The invention creatively introduces hydrophobic polymerizable monomers into phospholipid molecules, and constructs a polymer stable bracket in the phospholipid bilayer through ultraviolet crosslinking to improve the film forming stability of the phospholipid film.

Detailed Description

Example 1

A phospholipid bilayer membrane for nanopore sequencing is prepared by the following steps:

s1: taking the polycarbonate pretreated by SU-8 as a nanopore substrate, and carrying out fluorination modification treatment on the polycarbonate by using perfluorooctyl trichlorosilane to obtain a nanopore substrate with stronger hydrophobicity;

s2: the free radical inhibitors in EHMA and TEGDMA were removed by passing through an alumina column, and then, they were mixed with 2, 2-diethoxy-1-phenylhexanone (DEAP), a photoinitiator, in a molar ratio of 1:1:1, to obtain a monomer mixture, which was then mixed with an equimolar amount of diphytylphosphatidylcholine (DPhPC) amphiphatic phospholipid molecules and dissolved in physiological saline to obtain a monomer-doped amphiphatic phospholipid molecule solution.

S3: smearing the monomer-doped amphiprotic phospholipid molecule solution on the modified nano-pores, and drying by using nitrogen gas;

s4: adding 1mL of buffer solution to the two sides of each nanopore to redissolve the phospholipid molecules coated on the nanopore;

s5: absorbing the amphiphilic phospholipid molecular solution doped with the trace monomers by using a liquid-transferring gun to recoat the periphery of the nanopore to form a membrane;

s6: and (3) carrying out UV irradiation on the phospholipid membrane at a position 1-2cm away from the phospholipid bilayer membrane for 30min to ensure that the monomers distributed in the phospholipid sheet layer are subjected to photocrosslinking to form a polymer scaffold inside the phospholipid membrane.

S7: adding 2 mu L of alpha-HL solution into the buffer solution at one side of the nanopore, and then applying a bias potential of +40mV to the two sides of the phospholipid membrane to embed protein into the cross-linked phospholipid membrane to obtain the novel cross-linked phospholipid bilayer membrane with the protein channel.

Example 2

Example 2 is different from example 1 in that: DPhPC: EHMA: TEGDMA: the molar ratio of DEAP is 1: 0.5: 0.5: 0.5.

example 3

Example 3 is different from example 1 in that: DPhPC: EHMA: TEGDMA: the molar ratio of DEAP is 1: 2: 2: 2.

example 4

Example 4 is different from example 1 in that: the irradiation crosslinking time with an ultraviolet lamp was 20 min.

Example 5

Example 5 differs from example 1 in that: the irradiation crosslinking time with an ultraviolet lamp was 40 min.

Example 6

Example 6 differs from example 1 in that: gramicidin A was selected as a transmembrane protein to be embedded in phospholipid membranes.

Example 7

Example 7 is different from example 1 in that: before ultraviolet irradiation crosslinking, 2 mu L of alpha-HL solution is added into the buffer solution on one side of the nanopore, a bias potential of +40mV is applied to two sides of the phospholipid membrane to embed protein into the non-crosslinked phospholipid membrane, and then irradiation crosslinking is carried out for 30min by using an ultraviolet lamp.

Phospholipid membranes having protein channels were prepared under different conditions, and their breakdown voltages and phospholipid membrane lifetimes were measured, and the results are shown in Table 1. In table 1, DPhPC is diphytanoylphosphatidylcholine; TEGDMA is triethylene glycol dimethacrylate; EHMA is butyl methacrylate.

TABLE 1 stability of phospholipid membranes with protein channels prepared under different conditions

As can be seen from Table 1, the phospholipid membranes obtained by the method have poor stability and a service life of only about 6 hours, and the phospholipid membranes obtained by the method have greatly improved stability, the service life of the phospholipid membranes can be prolonged to 45 hours at most, and the breakdown voltage of the phospholipid membranes is improved to 2000 mV.

While the present invention has been described in detail with reference to the specific embodiments thereof, it should not be construed as limited by the scope of the present patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.